药食同源物质蜂蜜急性毒性和致突变性研究

Study on acute toxicity and mutagenicity of homologous functional food honey

目的 通过体内、体外及模式生物等方法,初步研究蜂蜜的急性毒性和潜在的致突变性。方法 采用小鼠霍恩氏法、CCK-8(细胞计数试剂)法和秀丽隐杆线虫3种模式,对蜂蜜进行急性毒性研究。通过组合实验(哺乳动物红细胞微核试验、体外哺乳类细胞TK基因突变试验和细菌回复突变试验)研究蜂蜜的致突变性。结果 蜂蜜小鼠急性经口毒性半数致死剂量LD_(50)及95%可信区间为雌性小鼠LD_(50)92.6 g/kg·bw(63.6~135.0 g/kg·bw);雄性小鼠LD_(50)92.6 g/kg·bw(57.0~150.0 g/kg·bw);蜂蜜人肾上皮细胞系293(HEK293)细胞急性毒性半数致死浓度LC50为464.2 g/L;秀丽隐杆线虫急性半数致死浓度LC50为176.9 g/L。7.5、15.0和30.0 g/kg·bw蜂蜜剂量组的小鼠骨髓嗜多染红细胞微核率与阴性对照组相比,差异均无统计学意义(χ^(2)=0.22,χ^(2)=0.00,χ^(2)=0.39,χ^(2)=0.18,χ^(2)=0.00,χ^(2)=0.00,P>0.05);1.25、2.50和5.00 mg/ml蜂蜜处理小鼠淋巴瘤细胞(L5178Y)总突变频率(T-MF值)与阴性对照组相比未见明显增加,增加倍数均在1倍以内;在加与不加S9混合液的条件下,8、40、200、1 000和5 000μg/皿5个浓度组的5种菌株(TA97a、TA98、TA100、TA102和TA1535)的回复突变菌落数与自发回复突变菌落数相近,致变比(MR值)均小于2。结论 依据食品安全国家标准,蜂蜜急性毒性分级判定为实际无毒;哺乳动物红细胞微核试验、体外哺乳类细胞TK基因突变试验和细菌回复突变试验均为阴性;应用体外细胞及秀丽隐杆线虫,初步得到急性毒性参考值;在本实验条件下,药食同源物质蜂蜜未见明显的急性毒性及致突变性。

Objective In order to evaluate the acute toxicity and mutagenicity of honey,in vivo,in vitro and model biology tests wereconducted.Methods The acute toxicity of honey was studied using Hohn's method,cell counting kit-8(CCK-8)assay and C.elegans.The mutagenicity of honey was assessed by a combination of mammalian erythrocyte micronucleus test,TK gene mutation test and bacterial reverse mutation assay.Results Median lethal dose(LD_(50))of acute oral toxicity of honey in female mice was 92.6 g/kg·bw(95%CI:63.6-135.0 g/kg·bw),and in male mice was 92.6 g/kg·bw(95%CI:57.0-150.0 g/kg·bw).Median lethal concentration(LC50)of acute toxicity of honey in human embryonic kidney 293 cells(HEK 293)was 464.2 g/L,and in C.elegans was 176.9 g/L.The micronucleus rates in 7.5,15.0,30.0 g/kg·bw dose were not significantly different from the negative control(χ^(2)=0.22,X2=0.00,χ^(2)=0.39,X2=0.18,χ^(2)=0.00,χ^(2)=0.00,P>0.05).The total mutation frequency(T-MF)of L5178Y mouse lymphoma cells treated with 1.25,2.50 and 5.00 mg/ml honey was not significantly increased compared with the negative control group.No increase in the number of revertant colonies was found in the bacterial reverse mutation assay with or without S9 mixture in 8,40,200,1000,5000μg/plate of honey in 5 testing Salmonella typhimurium stains TA97a,TA98,TA100,TA102 and TA1535.Conclusion According to the national standard of food safety,the acute toxicity classification of honey was judged to be actual non-toxic,mammalian erythrocyte micronucleus test,TK gene mutation test and bacterial reverse mutation assay were negative.The reference values of acute toxicity were obtained by using in vitro cells and C.elegans.No acute toxicity and mutagenicity were found in homologous functional food honey under the certain conditions of this study.